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Objective: Bridging the gap between experimental stroke and patients by ischemic blood probing during the hyperacute stage of vascular occlusion is crucial to assess the role of inflammation in human stroke and for the development of adjunct treatments beyond recanalization.Methods: We prospectively observed 151 consecutive ischemic stroke patients with embolic large vessel occlusion of the anterior circulation who underwent mechanical thrombectomy. In all these patients, we attempted microcatheter aspiration of 3 different arterial blood samples: (1) within the core of the occluded vascular compartment and controlled by (2) carotid and (3) femoral samples obtained under physiological flow conditions. Subsequent laboratory analyses comprised leukocyte counting and differentiation, platelet counting, and the quantification of 13 proinflammatory human chemokines/cytokines.Results: Forty patients meeting all clinical, imaging, interventional, and laboratory inclusion criteria could be analyzed, showing that the total number of leukocytes significantly increased under the occlusion condition. This increase was predominantly driven by neutrophils. Significant increases were also apparent for lymphocytes and monocytes, accompanied by locally elevated plasma levels of the T-cell chemoattractant CXCL-11. Finally, we found evidence that short-term clinical outcome (National Institute of Health Stroke Scale at 72 hours) was negatively associated with neutrophil accumulation.Interpretation: We provide the first direct human evidence that neutrophils, lymphocytes, and monocytes, accompanied by specific chemokine upregulation, accumulate in the ischemic vasculature during hyperacute stroke and may affect outcome. These findings strongly support experimental evidence that immune cells contribute to acute ischemic brain damage and indicate that ischemic inflammation initiates already during vascular occlusion. Ann Neurol 2020;87:466-479. [ABSTRACT FROM AUTHOR]

Now that mechanical thrombectomy has substantially improved outcomes after large-vessel occlusion stroke in up to every second patient, futile reperfusion wherein successful recanalization is not followed by a favorable outcome is moving into focus. Unfortunately, blood-based biomarkers, which identify critical stages of hemodynamically compromised yet reperfused tissue, are lacking. We recently reported that hypoxia induces the expression of endoglin, a TGF-β co-receptor, in human brain endothelium in vitro. Subsequent reoxygenation resulted in shedding. Our cell model suggests that soluble endoglin compromises the brain endothelial barrier function. To evaluate soluble endoglin as a potential biomarker of reperfusion (-injury) we analyzed its concentration in 148 blood samples of patients with acute stroke due to large-vessel occlusion. In line with our in vitro data, systemic soluble endoglin concentrations were significantly higher in patients with successful recanalization, whereas hypoxia alone did not induce local endoglin shedding, as analyzed by intra-arterial samples from hypoxic vasculature. In patients with reperfusion, higher concentrations of soluble endoglin additionally indicated larger infarct volumes at admission. In summary, we give translational evidence that the sequence of hypoxia and subsequent reoxygenation triggers the release of vasoactive soluble endoglin in large-vessel occlusion stroke and can serve as a biomarker for severe ischemia with ensuing recanalization/reperfusion. [ABSTRACT FROM AUTHOR]

Ischemic stroke caused by thromboembolic occlusion of large cerebral arteries, such as the internal carotid (ICA) and/or the middle cerebral artery (MCA), is treated by mechanical thrombectomy (MT). MT allows salvage of the vessel-occluding thrombemboli, which most frequently originate from the left atrium or the left ventricle of the heart or from sites of plaque rupture within large arteries above the heart. Clot composition may influence the efficacy of (intravenous) thrombolysis and MT, respectively. We analyzed 37 human thrombemboli obtained from acute ischemic stroke patients during MT with special emphasis on histological staining of neutrophils and neutrophil extracellular traps (NETs). We found neutrophils as the main cellular component of cerebral thrombemboli but encountered considerable morphological heterogeneity. Neutrophils accumulated in the border region of fibrin-rich structures indicating possible interaction of neutrophils with distinct structural thrombembolus components. Web-like NETs were found in 35 of 37 thrombemboli in varying amounts. NETs were almost exclusively found within fibrin-rich areas. Importantly, stroke etiology, age and present oral anticoagulation was associated with morphological patterns and the amount of neutrophils. Correlation of histological data and imaging data revealed that relative Hounsfield units of cerebral thrombemboli positively correlated with the amount of red blood cells. In summary, our results demonstrate that neutrophils and NETs are substantial constituents of cerebral thrombemboli and contribute to their structural complexity. [ABSTRACT FROM AUTHOR]

We aimed to assess the prognostic value of serum β-synuclein (β-syn), neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in patients with moderate-to-severe acute ischemic stroke. We measured β-syn, GFAP and NfL in serum samples collected one day after admission in 30 adult patients with moderate-to-severe ischemic stroke due to middle cerebral artery (MCA) occlusion. We tested the associations between biomarker levels and clinical and radiological scores (National Institute of Health Stroke Scale scores, NIHSS, and Alberta Stroke Program Early CT Score, ASPECTS), as well as measures of functional outcome (modified Rankin Scale, mRS). Serum biomarkers were significantly associated with ASPECTS values (β-syn p = 0.0011, GFAP p = 0.0002) but not with NIHSS scores at admission. Patients who received mechanical thrombectomy and intravenous thrombolysis showed lower β-syn (p = 0.029) und NfL concentrations (p = 0.0024) compared to patients who received only mechanical thrombectomy. According to median biomarker levels, patients with high β-syn, NfL or GFAP levels showed, after therapy, lower clinical improvement (i.e., lower 24-h NIHSS change), higher NIHSS scores during hospitalization and higher mRS scores at 3-month follow-up. Elevated serum concentrations of β-syn (p = 0.016), NfL (p = 0.020) or GFAP (p = 0.010) were significantly associated with 3-month mRS of 3–6 vs. 0–2 even after accounting for age, sex and renal function. In patients with moderate-to-severe acute ischemic stroke, serum β-syn, NfL and GFAP levels associated with clinical and radiological scores at different timepoints and were able to predict short- and middle-term clinical outcomes. [ABSTRACT FROM AUTHOR]

In stroke patients, local sampling of pial blood within the occluded vasculature before recanalization by mechanical thrombectomy emerged as powerful tool enabling insights into ultra-early stroke pathophysiology. Thereby, a strong intravascular inflammatory response hallmarked by hyper-acute neutrophil recruitment, altered lymphocyte composition and platelet activation could be observed. These human findings mirror experimental stroke. Here, neutrophil and T-cell activation are driven by platelets involving engagement of platelet glycoprotein receptor (GP)Ib, GPVI and CD84 as well as α-granule release orchestrating infarct progression. Thus, targeting of early intravascular inflammation may evolve as a new therapeutic strategy to augment the effects of recanalization. [ABSTRACT FROM AUTHOR]

In acute stroke, neuroinflammation can nowadays be analyzed by local cerebral aspiration of pial-ischemic blood during mechanical thrombectomy. Recently, Shaw et al. reported on differences in leukocyte subpopulations within the occluded cerebrovascular compartment. In their study, a main proportion of granulocytes was lost during isolation. By immediate analysis, we found a reproducible increase in absolute local granulocytes without variations in absolute lymphocyte and monocyte numbers. Flow-cytometric phenotyping confirmed a high proportion of granulocytes and a local shift towards CD4+ T cells. Thus, immediate analysis appears to be critical to observe distinct local responses of leukocytes to acute ischemic stroke. [ABSTRACT FROM AUTHOR]

In acute ischemic stroke due to large vessel occlusion (LVO) infarcts rapidly grow into the penumbra, which represents dysfunctional, but still viable brain tissue amenable to rescue by vessel recanalization. However, infarct progression and/or delayed patient presentation are serious and frequent limitations of this so far only acute therapy. Thus, a major goal of translational research is to "freeze" the penumbra already during LVO (before opening the vessel) and thereby extend individual time windows for non-futile recanalization. We used the filament occlusion model of the middle cerebral artery (MCAO) in mice and assessed progressive infarction under occlusion at 2, 3, and 4 h after onset. We show that blocking the activatory platelet receptor glycoprotein (GP)VI substantially delayed progressive neocortical infarction compared to isotype control antibody treated mice. Moreover, the local vascular recruitment of infiltrating neutrophils and T-cells was mitigated. In conclusion, our experimental data support ongoing clinical trials blocking platelet GPVI in acute ischemic stroke. • GPVI depletion delays progressive infarction during MCA occlusion. • Blocking GPVI reduces neuro-inflammation. • Targeting GPVI reduces neutrophil and T-cell recruitment to the ischemic brain. [ABSTRACT FROM AUTHOR]

Background: In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood. Methods: To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1−/− mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion. Results: We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion. Conclusions: Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization. [ABSTRACT FROM AUTHOR]

Background: In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood.Methods: To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1-/- mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion.Results: We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion.Conclusions: Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization. [ABSTRACT FROM AUTHOR]

High-mobility group box 1 protein (HMGB1) is a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis. NETs are regularly found in cerebral thromboemboli. We here analyzed associated HMGB1 expression in human thromboemboli retrieved via mechanical thrombectomy from 37 stroke patients with large vessel occlusion. HMGB1 was detected in all thromboemboli, accounting for 1.7% (IQR 0.6–6.2%) of the total thromboemboli area and was found to be colocalized with neutrophils and NETs and in spatial proximity to platelets. Correlation analysis revealed that the detection of HMGB1 was strongly related to the number of neutrophils (r = 0.58, p = 0.0002) and platelets (r = 0.51, p = 0.001). Our results demonstrate that HMGB1 is a substantial constituent of thromboemboli causing large vessel occlusion stroke. [ABSTRACT FROM AUTHOR]

It remains unclear if principal components of the local cerebral stroke immune response can be reliably and reproducibly observed in patients with acute large-vessel-occlusion (LVO) stroke. We prospectively studied a large independent cohort of n = 318 consecutive LVO stroke patients undergoing mechanical thrombectomy during which cerebral blood samples from within the occluded anterior circulation and systemic control samples from the ipsilateral cervical internal carotid artery were obtained. An extensive protocol was applied to homogenize the patient cohort and to standardize the procedural steps of endovascular sample collection, sample processing, and laboratory analyses. N = 58 patients met all inclusion criteria. (1) Mean total leukocyte counts were significantly higher within the occluded ischemic cerebral vasculature (I) vs. intraindividual systemic controls (S): +9.6%, I: 8114/µL ± 529 vs. S: 7406/µL ± 468, p = 0.0125. (2) This increase was driven by neutrophils: +12.1%, I: 7197/µL ± 510 vs. S: 6420/µL ± 438, p = 0.0022. Leukocyte influx was associated with (3) reduced retrograde collateral flow (R2 = 0.09696, p = 0.0373) and (4) greater infarct extent (R2 = 0.08382, p = 0.032). Despite LVO, leukocytes invade the occluded territory via retrograde collateral pathways early during ischemia, likely compromising cerebral hemodynamics and tissue integrity. This inflammatory response can be reliably observed in human stroke by harvesting immune cells from the occluded cerebral vascular compartment. [ABSTRACT FROM AUTHOR]

Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2−/− mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke. [ABSTRACT FROM AUTHOR]